Equine infectious anemia virus (EIAV) is a lentivirus distantly related to the human and simian immunodeficiency viruses (HIV and SIV). Our aim is to identify and characterize the cis-acting elements that respond to both cellular and viral transcription factors to regulate EIAV gene expression. Our first aim was to define the cis-acting element that mediates the transcriptional activation by the viral Tat protein. We have cloned the EIAV promoter sequences located in the viral long terminal repeat (LTR) in a plasmid upstream of the chloramphenicol acetyltransferase (CAT) gene. Promoter activity was quantified by analysis of CAT mRNA or CAT enzyme activity. The EIAV Tat-response element (TAR) was shown to be located in the viral LTR between positions +1 and +25 with respect to the RNA start site. Insertion of EIAV TAR in place of the HIV-1 TAR element within the HIV-1 promoter conferred responsiveness to EIAV Tat. Mutagenesis of individual or clusters of nucleotides within the EIAV TAR sequence revealed that it functions as an RNA stem-loop structure similar to the TAR element of HIV-1. Mutations that disrupted the hairpin structure abolished trans-activation; compensatory mutations that restored the hairpin but altered the primary sequence restored activity. Mutagenesis of specific nucleotides in the loop region had varied effects, some positions were tolerant of substitutions whereas others impaired function. Thus, the EIAV Tat protein specifically recognizes a U-G base pair positioned between the helical stem and the loop regions of TAR RNA and may interact with several loop nucleotides. An analysis of cis-acting DNA sequence elements that bind cellular transcription factors has been initiated.